Spinal muscular atrophy is one of the most common and severe autosomal recessive diseases with an overall incidence of about 1 in 6,000 to 10,000 live births and a carrier frequency of 1 in 35 to 1 in 117, depending on ethnicity. The disease is characterized by the progressive degeneration and loss of anterior horn cells in the spinal cord and brain stem nuclei causing symmetric muscle weakness and atrophy, and the clinical subtypes are primarily based on age at onset. Type I disease (Werdnig-Hoffmann disease, MIM#253300), which occurs in 60-70% of patients, is characterized by onset of respiratory insufficiency at birth or before six months of age leading to death within two years. Type I patients never sit or walk. Onset of Type II disease (MIM#253550) typically occurs after 6 months of age; these infants can sit, but never walk unaided, and have significantly reduced life expectancy. Patients with Type III disease (Kugelberg-Welander syndrome, MIM#253400) present after 18 months of age, stand and walk, but often become wheelchair-bound during childhood or early adulthood. Type IV disease (MIM#271150) is characterized by adult onset (mean age of 35 years) and slow disease progression.
Homozygous mutations of the SMN1 gene on chromosome 5q13.2 are the major cause of SMA (Lefebvre et al., Cell 80:155-165, 1995). In approximately 95-98% of SMA patients, both copies of SMN1 exons 7 and 8 are either deleted or are rendered non-functional due to gene conversion of SMN1 to SMN2 (Ogino et al., J. Hum. Genet. 12:1015-1023 (2004)). The remaining 2-5% of patients are compound heterozygotes that carry an intragenic mutation(s) on one allele and either a deletion or gene conversion mutation on the other allele (Wirth et al., Am J. Hum. Genet. 64:1340-1356, 1999). SMN1 and SMN2 are separated by approximately 1.4 Megabases and comprise the telomeric and centromeric members, respectively, of a set of genes, including NAIP, present within a segmental duplication on 5q13.2. The SMN1 (NCBI Reference Sequence NG_008691) and SMN2 genes have high sequence similarity even in the promoter regions and there are no encoded amino acid differences. A single base change affecting a putative splice enhancer in exon 7 (840C>T; position 27006 of SEQ ID NO:1), however, accounts for splicing differences such that the majority of SMN2 transcripts lack exon 7 (Lorson et al., Proc. Natl. Acad. Sci. (USA) 96:6307-6311 (1999)). Although some genotype/phenotype correlations have been established among patients that carry SMN1 point mutations, the presence of additional copies of SMN2 positively modifies clinical prognosis (Wirth et al., 1999).
The majority of mutations causing all four SMA types (i.e., Types I-IV) involve SMN1 copy number loss. Mutation detection typically involves PCR amplification of SMN1 exon 7, which is homozygously absent in most affected individuals. Carrier screening, however, is performed by dosage-sensitive but location-insensitive methods that can distinguish SMN1 and SMN2, but that provide no information on gene location. These carrier screens are currently performed using quantitative PCR (qPCR), Multiplex Ligation-Dependent Probe Amplification (MLPA), and/or Taqman quantitative technology. These methods cannot determine the number of SMN1 copies present on individual chromosomes. Given the tight linkage of the structurally related SMN1 and SMN2 genes and the opportunity for gene conversion as well as gene duplication or deletion, it is unsurprising that these methods exhibit detectability varying from 71-94% due to the inability to identify individuals that are SMA silent carriers, such as silent (2+0) carriers. Information on gene location is urgently needed to allow genetic counseling to consider the effects of segregation in germ cell production and its implications for offspring. Individuals with two SMN1 copies on one chromosome (a duplication allele) and no copies on the other (a deletion allele), are referred to as silent (2+0) carriers, as most individuals with two intact SMN1 copies, one copy on each chromosome (1+1), are not carriers (FIG. 1). Thus, SMA carrier detection by current techniques that are location-insensitive generates false negative results.
The frequency of silent (2+0) carriers varies based on ethnicity and is directly proportional to the product of the frequency of deletion and duplication alleles in a given population. Among Ashkenazi Jews, the SMA carrier frequency has been reported at 1 in 41 with a detectability of approximately 90%. Of the remaining 10%, about 8% are silent (2+0) carriers and the rest carry intragenic mutations. The frequency of SMN1 silent (2+0) carriers in any population modifies the risk of being a carrier after a negative screening result. For example, in the African American (AA) population, the frequency of duplication (2+1) individuals is relatively high (47%), and consequently there are more silent (2+0) carriers, such that the carrier detection rate is only 71% with a residual risk of 1 in 121 after a negative result.
Accordingly, a need exists in the art for screening methods for SMA and SMA carriers that are location-sensitive in addition to being sensitive to copy number, e.g., copy numbers of SMN1 and/or SMN2 alleles. An accurate assessment of the risk of producing offspring with SMA requires knowledge of both the copy numbers of SMN1 and/or SMN2 and of the location of those alleles that are found in a given genome. Such information is vital to competent genetic counseling of prospective parents.